Project Summary Robust HIV-specific CD4 T cell responses are associated with decreased viral load, an increased antibody neutralization breadth, and a slower progression to AIDS, and should therefore be considered in the development of a protective HIV vaccine. CD4 T cells are activated by antigen-derived peptides (epitopes) displayed in complex with MHC Class II molecules (MHCII) on the surface of an antigen presenting cell (APC). According to the standard model, internalized antigens are catabolized (?processed?) and loaded onto MHCII in late endosomal compartments. However, many alternative pathways have been described. These include the recycling pathway, in which peptide is loaded onto MHCII in the early endosome, and endogenous processing, which occurs when virally-derived antigens synthesized within infected APCs are proteolyzed and loaded onto MHCII via a complex network of intracellular pathways. While some HIV-1 epitopes can be presented from inactivated virus, others have been shown to be presented endogenously. However, the relative contributions of classical and alternative pathways to the HIV-specific CD4 T cell response are not known. Additionally, our understanding of the cell types that act as APCs during HIV-1 infection, and their relative contributions to CD4 T cell activation, is incomplete. Of note, in addition to the ?professional? APCs that present peptide via MHCII, CD4 T cells, which transiently express MHCII upon stimulation and are host cells for HIV, might act in this capacity. Accordingly, I will investigate the abilities of human dendritic cells, macrophages, and CD4 T cells to present HIV-1 epitopes from infectious and inactivated virus, and elucidate the cellular pathways utilized by these APCs for peptide production and MHCII loading. In Aim 1, I will define the differential abilities of dendritic cells, macrophages, and CD4 T cells to present epitopes from live and inactivated HIV-1 using established in vitro antigen presentation assays. In Aim 2, I will elucidate the cellular mechanisms used by DCs, macrophages, and CD4 T cells to present HIV antigens via MHC-II using siRNA and chemical inhibitors targeting key aspects of the antigen processing machinery. This work will provide insights into the pathways used by APCs to activate CD4 T cells in the context of an HIV-1 infection and has the potential to inform rational vaccine design. This project will allow me to develop into a confident, independent researcher. This training will expand my knowledge of cell biology and immunology and help me master valuable new technical skills. Additionally, I will improve my communication skills, allowing me to effectively communicate the importance of biomedical research with the public. Most importantly, this training will help me achieve my long- term professional goal of being an independent senior scientist.